In Drosophila, the homeobox gene tinman is absolutely required for development of the heart. In vertebrates, a small family of homeobox genes that are related to tinman have been identified, including tissues, their precise role in regulation of cardiac development remains unclear. This is illustrated by experiments investigators the role of the Nkx2-5 gene in cardiogenesis. Although, ablation of the Nkx2-5 gene in mouse results in embryonic death due to heart defects, the embryos contain a differentiated superficially normal , superficially normal heart and the major observable defect is a failure of looping morphogenesis. The role of Nk2-5 therefore, is not obviously similar to that of the tinman gene in the fly. The relatively minor consequences of Nkx-25 ablation, could be explained by the presence of additional tinman family genes with redundant activities, but at present the characterization of candidate rescuing genes is very preliminary. Therefore, the importance of the tinman gene family for vertebrate heart development is currently an unresolved question. In order to further explore the role of the tinman genes during heart development, we have carried out a series of experiments in the frog, Xenopus laevis. First, we have identified a novel member of the tinman family that is expressed early during heart development. Second we have carried out experiments in which expression of a dominant inhibitory mutant form of Nkx2-5 in the frog embryos results in total elimination of detectable myocardial differentiation. We proposed that this effect is achieved by inhibition of the function of multiple members of the tinman gene family. The long term objective of the proposed research is to understand the mechanism by which the vertebrate tinman-related genes regulate cardiac development. The specific experimental aims are as follows: -1) to further examine the mechanism leading to the elimination of myocardial gene expression. 2) to use a hormone-inducible expression systems to determine the time during development that Nkx2-5 plays its role in regulation of myocardial differentiation. 3). To determine whether Nkx2- 5, in combination with GATA-4 and SRF is sufficient to direct myocardial gene expression. 4) To identify the complete repertoire of tinman family members expressed during myocardial development of tinman family members expressed during myocardial development in Xenopus.